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theses

Histones are subject to various covalent post-translational modifications that can influence specific changes in chromatin structure and levels of transcription. The Polycomb (PcG) and Trithorax (TrxG) group systems are classic epigenetic systems that mediate transcriptional repression and activation, respectively, through mutually-exclusive modifications of histone H3 at lysine 27 (H3K27) in the gene promoter region. The Polycomb Repressive Complex 2 (PRC2) mediates transcriptional silencing by trimethylating H3K27 while TrxG proteins mediate transcriptional activation by acetylating H3K27. Histone deacetylases (HDACs) 1 and 2 are associated with the PRC2 complex and their overexpression has been linked to a variety of cancers. The Rabson Laboratory adapted SW480 colon cancer cells to the pan-HDAC inhibitor vorinostat. Vorinostat-adapted cells (SH80) showed a near elimination of tumorigenicity in tumor xenografts, global changes in mRNA expression, and an upregulation of PRC2 target genes. The overarching goal of this research was to determine which changes were necessary to generate the non-malignant phenotype of SH80. First, we used H3K27 trimethylation and acetylation status to evaluate changes at PRC2 targets as a function of i) vorinostat concentration and ii) acute treatment versus adaptation with vorinostat. Second, we investigated if selective inhibition of HDACs 1, 2, and 3 would be sufficient to generate the non-malignant phenotype by adapting SW480 cells to the inhibitor CI-994 and evaluating changes in proliferation, colony formation, and gene expression at PRC2 targets. Three PRC2 target genes were identified that had striking changes in mRNA expression as a function of vorinostat concentration and/or adaptation that correlated with H3K27 modifications and may be important to generating the non-malignant phenotype: TSPAN8, STMN2, and EDN3. Cells adapted to 12 µM CI-994 showed a substantial reduction in colony formation, suggesting that targeted inhibition of HDACs 1, 2, and 3 may reduce tumorigenicity. Changes in mRNA expression in CI-994 adapted cells were similar to SH80 cells, indicating that inhibition of HDACs 1, 2, and 3 leads to altered expression of PRC target genes. Substantial changes in gene expression were observed for TSPAN8, STMN2, and EDN3, further reinforcing the idea that these genes may be of particular importance to the non-malignant phenotype.

ETD_7201

M.S.

Includes bibliographical references

by Diana S. Vengsarkar

doi:10.7282/T3QN68Z9

ETD graduate

The author owns the copyright to this work.